Method for preparing a sample solution containing neurogranin-related peptides and method for analyzing neurogranin-related peptides.

Abstract

Provided are: a method which is for analyzing a neurogranin-related peptide and can suppress deviations in analysis results; and a method for preparing a biological sample containing the neurogranin-related peptide used for same. A method for preparing a sample solution containing a neurogranin-related peptide involves mixing a biological sample containing the neurogranin-related peptide with an organic solvent having a relative polarity of 0.200 to 0.700 to prepare a sample solution containing the organic solvent having a final concentration of at least 5.0 (v / v)%.

Description

Technical Field

[0001] The present invention relates to a method for preparing a sample solution containing a neurogranin-related peptide and a method for analyzing a neurogranin-related peptide.

Background Art

[0002] Alzheimer's disease is the main type of dementia, and the number of its sufferers has been increasing in recent years, and its research has become even more important. The onset of Alzheimer's disease is deeply related to Aβ-related peptides such as amyloid β (Aβ) generated by cleavage of amyloid precursor protein (APP; Amyloid precursor protein). And it has been reported that a plurality of Aβ-related peptides in blood can be detected by combining immunoprecipitation and mass spectrometry, and the detected specific ratio of Aβ-related peptides can be effectively used as a blood biomarker for cerebral amyloid accumulation (Non-Patent Documents 1 to

[0003] 2, Patent Documents 1 to 3).

[0003] On the other hand, in order to monitor the progression of the pathological condition of Alzheimer's disease, various biomarkers are required. In addition to amyloid accumulation, biomarkers for reflecting each process such as tau accumulation and neurodegeneration are required. Among them, neurogranin is one of the biomarkers for neurodegeneration, and it has been reported that it increases in the cerebrospinal fluid (CSF) of Alzheimer's disease patients (Non-Patent Documents 3, Non-Patent Documents 4). Further, it has also been reported that fragmentation of neurogranin is promoted in the brain of Alzheimer's disease patients, and the fragment peptides thereof are present in blood, and furthermore, translated and modified neurogranin such as acetylation or glutathionylation is also present (Non-Patent Document 5). Therefore, by detecting neurogranin-related peptides such as neurogranin or its fragment peptides from biological samples such as blood and CSF, a means for confirming neurodegeneration is expected.

Prior Art Documents

Patent Documents

[0004] [License 1] WO2015 / 178398 [License 2] WO2017 / 47529 [License 3] Announcement No. 2017-20980 [Non-licensed literature]

[0005] [Non-licensed Document 1] Kaneko N, Nakamura A, Washimi Y, Kato T, Sakurai T, Arahata Y, Bundo M, Takeda A, Niida S, Ito K, Toba K, Tanaka K, Yanagisawa K. : Novel plasma biomarker surrogating cerebral amyloid deposition. Proc Jpn Acad Ser B Phys Biol Sci. 2014;90(9):353-364. [Non-licensed Document 2] : High performance plasma amyloid-β biomarkers for Alzheimer's disease. Nature. 2018;554(7691):249-254. [Non-licensed Document 3] Portelius E, Olsson B, Hoglund K, Cullen NC, Kvartsberg H, Andreasson U, Zetterberg H, Sandelius A, Shaw LM, Lee VMY, Irwin DJ, Grossman M, Weintraub D, Chen-Plotkin A, Wolk DA, McCluskey L, Elman L, McBride J, Tolenowski JB, Tronowski JB, Tronowski JQ K. : Cerebrospinal fluid neurogranin concentration in neurodegeneration: relation to clinical phenotypes and neuropathology. Acta Neuropathol. 2018 ;136(3):363-376.

Fashion 4

Wood 5

[0006] Incidentally, blood samples are preferred as biological samples for analyzing neurolanin-related peptides because they can be collected through general examinations and are minimally invasive. However, because neurolanin-related peptides present in blood and other biological samples are present in trace amounts, depending on the analytical method, a problem can arise where the values ​​of the analysis results vary greatly even from the same blood sample.

[0007] The present invention aims to provide a method for analyzing neurogranin-related peptides that can suppress variability in analytical results, and a method for preparing a biological sample containing neurogranin-related peptides for use therein. [Means for solving the problem]

[0008] The first aspect of the present invention relates to a method for preparing a neurogranin-related peptide-containing biological sample, which involves mixing a biological sample containing a neurogranin-related peptide with an organic solvent having a relative polarity of 0.200 to 0.700 to prepare a sample solution in which the final concentration of the organic solvent is 5.0 (v / v) or higher.

[0009] The analytical method according to the first embodiment of the present invention, using the preparation method according to the first embodiment, is an analytical method for neurogranin-related peptides comprising a preparation step of carrying out the preparation method according to the first embodiment, and a measurement step of performing mass spectrometry, liquid chromatography, immunoassay, or surface plasmon resonance using the sample solution. [Effects of the Invention]

[0010] According to the preparation method of the first aspect of the present invention, the degradation of neurogranin-related peptides contained in biological samples can be suppressed. Furthermore, according to the analysis method of the first aspect of the present invention, variability in the analysis results of neurogranin-related peptides can be suppressed. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the mass spectra (final concentrations 0-5%) of Ng-related peptide-containing sample solutions at different DMSO concentrations. The vertical axis represents relative intensity, and the horizontal axis represents m / z. [Figure 2] Figure 2 shows the mass spectra (concentrations 0-30%) of a sample solution containing Ng-related peptides at different DMSO concentrations. [Figure 3] Figure 3 is a graph showing the relationship between the ratio of SIL-Ng50-75 to SIL-Ng50-78 and the DMSO concentration (final concentration 0-5%). The vertical axis represents the ratio of SIL-Ng50-75 to SIL-Ng50-78, and the horizontal axis represents the final concentration of DMSO. [Figure 4] Figure 4 is a graph showing the relationship between the ratio of SIL-Ng50-75 to SIL-Ng50-78 and DMSO concentration (final concentrations 0-30%). [Figure 5] Figure 5 is a graph showing the relationship between the ratio of rNg1-75 to rNg1-78 and the DMSO concentration (final concentration 0-5%). The vertical axis represents the ratio of rNg1-75 to rNg1-78, and the horizontal axis represents the final concentration of DMSO. [Figure 6] Figure 6 is a graph showing the relationship between the ratio of rNg1-75 to rNg1-78 and the DMSO concentration (final concentration 0-30%). [Figure 7] Figure 7 is a mass spectrum for each incubation time in a sample solution containing an Ng-related peptide of the comparative example. The vertical axis represents relative intensity, and the horizontal axis represents m / z. [Figure 8] Figure 8 is a graph showing the relationship between the ratio of SIL-Ng50-75 to SIL-Ng50-78 and the incubation time in the comparative example. The vertical axis represents the ratio of SIL-Ng50-75 to SIL-Ng50-78, and the horizontal axis represents the incubation time at room temperature. [Figure 9] Figure 9 is a graph showing the relationship between the ratio of rNg1-75 to rNg1-78 and the incubation time in the comparative example. The vertical axis represents the ratio of rNg1-75 to rNg1-78, and the horizontal axis represents the incubation time at room temperature. [Figure 10] Figure 10 is a graph showing the relationship between the ratio of SIL-Ng50-75 to SIL-Ng50-78 and various solvents. The vertical axis represents the ratio of SIL-Ng50-75 to SIL-Ng50-78, and the horizontal axis represents various solvents (final concentration 10%). [Figure 11] Figure 11 is a graph showing the relationship between the ratio of rNg1-75 to rNg1-78 and various solvents. The vertical axis represents the ratio of rNg1-75 to rNg1-78, and the horizontal axis represents various solvents (final concentration 10%). [Figure 12] Figure 12 is a mass spectrum for each ACN concentration (final concentration 0 to 20%) in a sample solution containing an Ng-related peptide. The vertical axis represents relative intensity, and the horizontal axis represents m / z. [Figure 13] Figure 13 shows an enlarged view in the vicinity of m / z 2150 to 2540 in FIG. 12. [Figure 14] Figure 14 shows an enlarged view in the vicinity of m / z 4800 to 5000 in FIG. 12.

Mode for Carrying Out the Invention

[0012] 1. First Embodiment 1-1. Preparation Method The method for preparing a neurogranin-related peptide-containing sample solution according to the first embodiment involves mixing a biological sample with an organic solvent having a relative polarity of 0.200 or higher and 0.700 or lower.

[0013] Furthermore, "neurogranin-related peptides" (hereinafter abbreviated as "Ng-related peptides") include neurogranin, translated and / or modified neurogranin, and their fragment peptides.

[0014] Biological samples are samples containing Ng-related peptides, and include, for example, bodily fluids such as blood, cerebrospinal fluid, urine, body secretions, saliva, sputum, and feces. Blood includes whole blood, plasma, serum, etc. Blood may be whole blood collected from an individual and then processed by centrifugation, freezing, etc. Blood is preferred. Blood is less invasive than cerebrospinal fluid and is readily available as a screening sample in health checkups, etc.

[0015] The relative polarity of organic solvents is between 0.200 and 0.700. The lower limit of relative polarity is preferably 0.300 or higher, more preferably 0.370 or higher, and most preferably 0.400 or higher. The upper limit is preferably 0.600 or lower, more preferably 0.500 or lower, and most preferably 0.450 or lower. Relative polarity is disclosed in detail, for example, in "Solvents and Solvent Effects in Organic Chemistry", Christian Reichards, Wiley-VCH Publishers, 3rd ed., 2003.

[0016] Examples of such organic solvents include acetone (0.355), dimethylformamide (0.386), dimethyl sulfoxide (0.444), acetonitrile (0.460), 2-propanol (0.546), ethanol (0.654), 1-butanol (0.586), and 2-butanol (0.506). The numbers in parentheses indicate the relative polarity. These may be used individually or in combination of two or more. From the viewpoint of more reliably suppressing the degradation of Ng-related peptides, dimethyl sulfoxide (DMSO) is preferred. On the other hand, from the viewpoint of suppressing the degradation of Ng-related peptides and detecting them with even higher sensitivity, acetonitrile (ACN) is preferred.

[0017] In the preparation step, the biological sample and the organic solvent are mixed so that the final concentration of the organic solvent is 5.0 (v / v)% or more relative to the prepared Ng-related peptide-containing sample solution. The lower limit of the final concentration is preferably 10.0 (v / v)% or more, and the upper limit is, for example, 30.0 (v / v)% or less, preferably 20.0 (v / v)% or less. By setting the concentration above the lower limit, the degradation of Ng-related peptide due to organic solvent mixing is effectively suppressed. Furthermore, by setting the concentration below the upper limit, the decrease in detection sensitivity of Ng-related peptide due to excessive addition of organic solvent can be suppressed. In addition, when acetonitrile is used, the concentration is preferably 15.0 (v / v)% or more and more preferably 20.0 (v / v)% or less, in addition to the above range. By using these concentration ranges, Ng-related peptide can be detected with higher sensitivity, so that, for example, the peaks of more types of Ng-related peptide can be detected in the mass spectrum obtained by the mass spectrometry method described later. Furthermore, the amount of organic solvent added per 100 volumes of biological sample is, for example, 5.0 volumes or more, preferably 10.0 volumes or more, and also, for example, 200 volumes or less, preferably 50.0 volumes or less.

[0018] In the preparation step, it is preferable to add a buffer solution as needed to achieve the above final concentration. Examples of buffer solutions include Tris buffer, phosphate buffer, HEPES buffer, and ammonium acetate buffer. This allows the pH of the sample solution to be neutralized to achieve the above final concentration and facilitates purification in the purification step described later. The pH of the buffer solution is preferably neutral, for example, pH 6.0 or higher, preferably 6.5 or higher, and also, for example, 8.5 or lower, preferably 8.0 or lower. The mixing ratio of the buffer solution is, for example, 5.0 (v / v)% or higher, preferably 10.0 (v / v)% or higher, and also, for example, 80.0 (v / v)% or lower, preferably 40.0 (v / v)% or lower, relative to the prepared Ng-related peptide-containing sample solution. Also, the amount of buffer solution is 50.0 parts by volume or higher, preferably 100 parts by volume or higher, and also, for example, 1000 parts by volume or lower, preferably 500 parts by volume or lower, relative to 100 parts by volume of organic solvent.

[0019] Furthermore, when the purification process described later is carried out continuously, the surfactant may be mixed with the biological sample together with the buffer solution described later in the first binding step.

[0020] This process prepares (manufactures) a sample solution containing Ng-related peptides. Because this sample solution contains a specific amount of a particular organic solvent, the degradation of Ng-related peptides in the sample solution can be suppressed. That is, even after storage for a predetermined time, the decrease in Ng-related peptide content over time is suppressed, and the amount of Ng-related peptides from the time of biological sample collection remains relatively constant. Therefore, even if measurement (described later) is performed after storage for a predetermined time, changes in detection concentration due to storage time are suppressed, and variations in detection concentration can be reduced. Furthermore, degradation occurring during the measurement process can also be suppressed. Therefore, changes in detection concentration due to the measurement process time are suppressed, and variations in detection concentration can be reduced.

[0021] The inventors focused on Ng-related peptides contained in biological samples such as blood collected from living organisms and discovered that Ng-related peptides are degraded (digested) over time in biological samples. Specifically, they found that the C-terminus of the 75th amino acid of neurogranin in Ng-related peptides is cleaved over time by proteases in the biological sample. That is, taking Ng1-78 as the target of analysis, when blood samples are collected (separated) from living organisms and analyzed, Ng1-78 is gradually degraded into Ng1-75 and Ng76-78 during the storage time or analysis preparation time from collection to analysis, and Ng1-78 changes (decreases) according to that time. As a result of further diligent investigation, it was found that the degradation of the amino acid is suppressed by presenting a specific amount of a specific organic solvent in the biological sample, and thus the preparation method of the present invention was completed. Furthermore, while Ng1-78 is constantly being broken down in living organisms, the amount of Ng1-78 produced by the organism is kept constant. Therefore, the aforementioned decrease in Ng1-78 only occurs from the moment it is separated from the organism and prepared as a biological sample for analysis.

[0022] 1-2.Analysis process The analytical method according to the first embodiment is a method for analyzing Ng-related peptides in a biological sample, comprising a preparation step and a measurement step in order. The preparation step is the preparation method described above, and the measurement step is to perform mass spectrometry. In a preferred form, a purification step is performed within the measurement step. That is, the measurement step comprises, in order, a purification step for purifying Ng-related peptides in a sample solution containing Ng-related peptides, and a detection step for performing mass spectrometry. This form will be described in detail below.

[0023] [Refining process] The purification process is preferably affinity purification. Affinity purification may be performed once or twice, but from the viewpoint of detecting Ng-related peptides with higher sensitivity, two purifications are preferably performed. In this case, the purification process comprises, in this order, a first binding step (an example of a binding step), a first washing step (an example of a washing step), a first elution step, a neutralization step, a second binding step, a second washing step, and a second elution step (an example of an elution step). Each step will be described in detail below.

[0024] (1st bonding process) In the first binding step, the Ng-related peptide-containing sample solution obtained in the preparation step is brought into contact with the first support. As a result, the Ng-related peptide in the sample solution binds to the first support, and the first conjugate is obtained.

[0025] The first support can be any material to which Ng-related peptides can be bound, such as an antibody immobilization support.

[0026] The antibody immobilized on the first carrier is an antibody having an antigen-binding site capable of recognizing Ng-related peptides (anti-Ng-related peptide antibody), and examples include immunoglobulins or fragments thereof having an antigen-binding site capable of recognizing Ng-related peptides.

[0027] Examples of immunoglobulins include IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgY, IgD, and IgE. Examples of immunoglobulin fragments include F(ab')2, F(ab'), F(ab), Fd, Fv, light chains, and heavy chains. More specifically, clones NG2, NG7, EPR21152, and their fragments. The antibody may be either a monoclonal antibody or a polyclonal antibody.

[0028] Examples of materials for the first carrier include agarose, cepharose, dextran, silica gel, polyacrylamide, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, (meth)acrylic acid polymers, fluororesins, metal complex resins, glass, metals, and magnetic materials.

[0029] The shape of the first carrier can be spherical (including bead-shaped), plate-shaped, needle-shaped, or irregular, and may also be the wall of a channel within a microdevice.

[0030] In this case, in addition to the first carrier, a binding solution may be added as needed. The binding solution is preferably a neutral buffer containing a surfactant. Examples of buffers include those similar to the buffer described above in the preparation step. The pH of the binding solution is, for example, pH 6.0 or higher, preferably 6.5 or higher, and also, for example, 8.5 or lower, preferably 8.0 or lower.

[0031] Examples of surfactants contained in the binding solution include neutral surfactants having 7 to 15 (preferably 9 to 11) carbon atoms in the hydrophobic group. This suppresses nonspecific adsorption to the first conjugate and reduces ionization interference in mass spectrometry. Examples of such surfactants include surfactants having maltose in the hydrophilic portion, such as n-nonyl-β-D-maltoside, n-nonyl-β-D-thiomaltoside, n-decyl-β-D-maltoside, and n-undecyl-β-D-maltoside (UDM: n-Undecyl-β-D-maltoside); surfactants having trehalose in the hydrophilic portion, such as α-D-glucopyranosylα-D-glucopyranoside monodecanoate (trehalose C10); and surfactants having glucose in the hydrophilic portion, such as n-decyl-β-D-glucoside. These surfactants can be used individually or in combination of two or more types.

[0032] The surfactant concentration in the binding solution is, for example, 0.01% (w / v) or more, preferably 0.05% (w / v) or more, and also, for example, 10% (w / v) or less, preferably 3% (w / v) or less. If the surfactant concentration is within the above range, micelles will be sufficiently formed, and the effect of the surfactant can be reliably exerted.

[0033] Before the first binding step, pretreatment such as removing antibodies like IgG and IgM may be performed as needed.

[0034] (First washing process) In the first washing step, the first conjugate is washed using the first washing solution after the first bonding step.

[0035] The first washing solution is preferably a neutral buffer containing a surfactant. This effectively removes highly hydrophobic unwanted components (such as blood proteins, lipids, and glycolipids). The neutral buffer and surfactant in the first washing solution are the same as those exemplified in the binding solution.

[0036] The surfactant concentration in the first washing solution is, for example, 0.01% (w / v) or more, preferably 0.02% (w / v) or more, and also, for example, 5% (w / v) or less, preferably 2% (w / v) or less. If the surfactant concentration is within the above range, micelles will be sufficiently formed, and the effect of the surfactant can be reliably exerted.

[0037] Any known cleaning method may be used, and preferably, cleaning is performed multiple times. For example, cleaning is performed using a neutral buffer containing a surfactant, followed by cleaning with a neutral buffer that does not contain a surfactant.

[0038] A neutral buffer solution that does not contain surfactants can be used, similar to the neutral buffer solution exemplified in the bonding solution. This suppresses foaming caused by surfactants remaining in the first bond.

[0039] For washing, any general method can be used, such as stirring the carrier in the washing solution or spraying the washing solution from a washing nozzle. After washing with these neutral buffer solutions, further washing with water may be performed as needed.

[0040] (1st elution step) In the first elution step, after the first washing step, the first conjugate is brought into contact with the first acidic solution. This causes the Ng-related peptide to dissociate from the first conjugate, and the Ng-related peptide is eluted into the first acidic solution. As a result, a first eluate containing the Ng-related peptide is obtained.

[0041] Examples of the first acidic solution include glycine buffer and acidic aqueous solutions such as hydrochloric acid, with glycine buffer being preferred. The pH of the first acidic solution is, for example, 3.5 or less, preferably 3.0 or less, and also, for example, 0.5 or more, preferably 1.0 or more.

[0042] The first acidic solution preferably contains a surfactant. This allows for more reliable dissociation of the Ng-related peptide from the first conjugate. It also suppresses the adhesion of the eluted Ng-related peptide to containers such as test tubes and microplates. Therefore, the recovery rate of the Ng-related peptide can be reliably improved, thereby enhancing detection sensitivity. The surfactant used in the first acidic solution is the same as the surfactant exemplified in the conjugate solution. The surfactant concentration in the first acidic solution is the same as the surfactant concentration in the first washing solution.

[0043] (Neutralization process) In the neutralization step, after the first elution step, the first eluate is mixed with a neutral buffer. This neutralizes the first eluate, yielding a purified solution containing Ng-related peptides.

[0044] The neutral buffer used in the neutralization step preferably contains a surfactant. This suppresses nonspecific adsorption to the second conjugate in the second bonding step. Examples of the neutral buffer and surfactant used in the neutralization step are the same as those exemplified in the bonding solution. Furthermore, the surfactant concentration in the neutral buffer is the same as the surfactant concentration in the first bonding solution.

[0045] The pH of the resulting purified solution is neutral, for example, pH 6.0 or higher, preferably 6.5 or higher, and for example, 8.5 or lower, preferably 8.0 or lower. This improves the bonding efficiency in the second bonding step.

[0046] (Second bonding process) In the second binding step, after the neutralization step, the purified solution is brought into contact with the second support. This causes the Ng-related peptide in the purified solution to bind to the second support, yielding the second conjugate.

[0047] Preferably, the second support is an antibody-immobilized support, and specifically, it is the same as the antibody-immobilized support exemplified in the first support.

[0048] (Second washing process) In the second washing step, the second bond is washed using the second washing solution after the second bonding step.

[0049] The second washing solution is preferably a neutral buffer containing a surfactant. This allows for the effective removal of unwanted components with high hydrophobicity (such as blood proteins, lipids, and glycolipids). The neutral buffer and surfactant used in the second washing solution are the same as those exemplified in the binding solution. The surfactant concentration in the second washing solution is the same as that in the first washing solution.

[0050] Any known cleaning method may be used; specifically, the same method as the cleaning method exemplified in the first cleaning step should be implemented.

[0051] (Second elution step) In the second elution step, after the second washing step, the second conjugate is brought into contact with the second acidic solution. This causes the Ng-related peptide to dissociate from the second conjugate, and the Ng-related peptide is eluted into the second acidic solution. As a result, a second eluate containing the Ng-related peptide is obtained.

[0052] Examples of acidic aqueous solutions constituting the second acidic solution include those similar to the first acidic solution exemplified in the first elution step, and hydrochloric acid is preferred.

[0053] The second acidic solution preferably contains a volatile organic solvent. This allows for efficient dissociation of the Ng-related peptide from the second conjugate and elution into the second acidic solution, thereby improving the recovery rate of the Ng-related peptide.

[0054] Examples of volatile organic solvents include organic solvents that are miscible with water in any proportion, such as acetonitrile, methanol, ethanol, acetone, toluene, isopropanol, hexane, butanol, cyclohexane, ethylene glycol, benzene, chloroform, acetaldehyde, triethylamine, phenol, naphthalene, formaldehyde, tetrahydrofuran, and ethyl acetate. Preferably, acetonitrile, methanol, ethanol, acetone, and isopropanol are used. These organic solvents can be used individually or in combination of two or more.

[0055] The concentration of the volatile organic solvent in the second acidic solution is, for example, 10% (v / v) or more, preferably 25% (v / v) or more, and also, for example, 90% (v / v) or less, preferably 80% (v / v) or less. If the concentration is within the above range, the Ng-related peptide can be efficiently dissociated from the second support, and the sensitivity (S / N ratio) during mass spectrometry can be improved.

[0056] The second acidic solution preferably further contains amino acids such as methiotine. This reduces the oxidation of Ng-related peptides between the time the solution is placed in the mass spectrometer and the start of analysis, thereby improving detection sensitivity. The amino acid concentration in the second acidic solution is, for example, 0.01 mM or higher, preferably 0.05 mM or higher, and also, for example, 5 mM or lower, preferably 1 mM or lower.

[0057] The second acidic solution preferably further contains a protein of 9 kDa or greater. This ensures that the mass spectrometry sample contains the above protein, which acts as a proton acceptor during mass spectrometry, efficiently ionizing Ng-related peptides and significantly improving the detection sensitivity of Ng-related peptides. The upper limit of the protein mass is, for example, 100 kDa or less, preferably 15 kDa or less. Such proteins are not limited to those with a mass of 9 kDa or greater, and specific examples include bovine serum albumin (BSA), cytochrome, ovalbumin, and lysozyme. The protein concentration in the second acidic solution is, for example, 10 nM or more, preferably 30 nM or more, and also, for example, 600 nM or less, preferably 150 nM or less.

[0058] [Detection process] In the detection step, after the purification step, mass spectrometry is performed on the second eluate to detect Ng-related peptides.

[0059] Examples of ionization methods in mass spectrometry include MALDI (Matrix-Assisted Laser Desorption / Ionization), ESI (Electrospray Ionization), and APCI (Atmospheric Pressure Chemical Ionization). From the viewpoint of being able to detect trace amounts of Ng-related peptides in the sample solution with high sensitivity, MALDI is preferred.

[0060] Examples of mass spectrometry methods include, but are not limited to, TOF-MS (time-of-flight mass spectrometry), IT-MS (ion-trap mass spectrometry), IT-TOF-MS (ion-trap-time-of-flight mass spectrometry), and FTICR-MS (Fourier transform ion cyclotron mass spectrometry). When MALDI is used as the ionization method, these mass spectrometry methods are collectively referred to as MALDI-MS.

[0061] The specific detection procedure using MALDI-MS involves, for example, first dropping a matrix-containing solution onto a MALDI plate and drying (crystallizing) it to create the matrix. Next, a second eluate is dropped onto the matrix and dried to obtain a MALDI-MS sample. Subsequently, the MALDI-MS sample is irradiated with a laser to ionize Ng-related peptides, and these ionized peptides are detected by the detector of the aforementioned apparatus and output as a mass spectrum. By analyzing this mass spectrum, analytical results such as the type and concentration of Ng-related peptides contained in the biological sample can be obtained.

[0062] Examples of matrices include α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid, sinapic acid, and 3-aminoquinoline. These matrices can be used individually or in combination of two or more.

[0063] Examples of solvents for containing the matrix include acetonitrile, trifluoroacetic acid, methanol, ethanol, and water. These solvents can be used individually or in combination of two or more. The matrix concentration in the matrix-containing solution is, for example, 0.1 mg / mL or more, preferably 0.5 mg / mL or more, and also, for example, 50 mg / mL or less, preferably 10 mg / mL or less.

[0064] Preferably, a matrix additive is used in combination with the matrix. Examples of matrix additives include phosphonic acid group-containing compounds and ammonium salts, and preferably, phosphonic acid group-containing compounds are used from the viewpoint of suppressing adverse effects on the background due to residual washing solution. Examples of phosphonic acid group-containing compounds include phosphonic acid, methylphosphonic acid, phenylphosphonic acid, 1-naphthylmethylphosphonic acid, methylenediphosphonic acid (MDPNA), ethylenediphosphonic acid, ethane-1-hydroxy-1,1-diphosphonic acid, nitrilotriphosphonic acid, and ethylenediaminotetraphosphonic acid. The concentration of the matrix additive in the matrix-containing solution is, for example, 0.01% (w / v) or more, preferably 0.1% (w / v) or more, and also, for example, 10% (w / v) or less, preferably 1% (w / v) or less.

[0065] This analytical method can suppress variability in the analysis results of Ng-related peptides. In particular, even when biological samples collected from living organisms are stored for a desired period of time, it can suppress variability in measured values ​​at different storage times (for example, variability in the peak intensities and ratios of various Ng-related peptides in mass spectra, and the concentrations calculated from them).

[0066] Based on the knowledge gained from the above preparation method, the inventors conducted further intensive studies and discovered that, when analyzing Ng-related peptides, if a specific amount of a specific organic solvent is pre-added to a biological sample to suppress the degradation of Ng-related peptides, and this solution is used as the measurement sample for analysis, variability in Ng peptide analysis caused by storage time or analysis preparation time can be suppressed. This led to the completion of the analytical method of the present invention.

[0067] 1-3. Variations In the above embodiment, mass spectrometry is performed as the detection step, but for example, liquid chromatography, immunoassay, or surface plasmon resonance may be performed as the detection step. These measurement methods can be carried out according to known or conventional methods, except that the Ng-containing sample solution prepared in the above preparation step is used as the measurement sample. From the viewpoint of reliably detecting even minute amounts of Ng-related peptides in the blood, it is preferable to perform mass spectrometry as the detection step.

[0068] 2. Appearance Those skilled in the art will understand that the above-described exemplary embodiments are specific examples of the following embodiments.

[0069] (Section 1) A method for preparing a sample solution containing neurogranin-related peptide according to one embodiment may be to mix a biological sample containing neurogranin-related peptide with an organic solvent having a relative polarity of 0.200 or more and 0.700 or less, to prepare a sample solution in which the final concentration of the organic solvent is 5.0 (v / v) or more.

[0070] (Section 2) In the preparation method of Section 1, the final concentration of the organic solvent may be 5.0 (v / v)% or more and 20.0 (v / v) or less.

[0071] (Clause 3) In the preparation method described in paragraph 1 or 2, the biological sample may be blood.

[0072] (Clause 4) In the preparation method described in any one of paragraphs 1 to 3, the biological sample may be further mixed with a buffer solution.

[0073] (Section 5) In the preparation method described in any one of paragraphs 1 to 4, the organic solvent may be dimethyl sulfoxide.

[0074] (Item 6) In the preparation method described in any one of items 1 to 4, the organic solvent may be acetonitrile.

[0075] (Section 7) A method for analyzing neurogranin-related peptides according to one embodiment may comprise a preparation step of carrying out the preparation method described in any one of Sections 1 to 6, and a measurement step of carrying out mass spectrometry, liquid chromatography, immunoassay, or surface plasmon resonance using the sample solution.

[0076] (Clause 8) In the analytical method described in Clause 7, the measurement step may include: (a) a binding step of contacting the sample solution with a carrier to obtain a conjugate in which the neurogranin-related peptide is bound to the carrier; (b) a washing step of washing the conjugate with a washing solution; (c) an elution step of contacting the conjugate with an acidic solution to obtain an eluate in which the neurogranin-related peptide has eluted into the acidic solution; and (d) a detection step of detecting the neurogranin-related peptide in the eluate by mass spectrometry.

[0077] (Section 9) In the analytical method described in Section 8, the ionization method of the mass spectrometry may be a matrix-assisted laser desorption ionization method. [Examples]

[0078] The present invention will now be described in detail with reference to examples, but the scope of the present invention is not limited thereto.

[0079] <Example 1> [Preparation process] A DMSO-containing buffer solution was prepared by mixing 50 μL of DMSO (dimethyl sulfoxide) with 200 μL of a surfactant-containing buffer solution (0.1% n-undecyl-β-D-maltoside (UDM), 800 mM GlcNAc, 100 mM Tris-HCl, 300 mM NaCl; pH 7.4) to a DMSO-containing buffer solution with a DMSO concentration of 20.0 (v / v)%. Subsequently, a Ng·DMSO-containing buffer solution was prepared by mixing this buffer solution with 50 pM of stable isotope-labeled Ng50-78 (SIL-Ng50-78; manufactured by Toray Research Center Co., Ltd.) and 6 nM of His-tagged recombinant Ng (rNg; manufactured by abcam Co., Ltd.). Note that in SIL-Ng50-78, the carbon atoms and nitrogen atoms of Pro and Val are respectively... 13 C and 15 It is replaced with N.

[0080] Next, 250 μL of commercially available human plasma sample was mixed with 250 μL of Ng·DMSO-containing buffer. This prepared a sample solution containing Ng-related peptides with a final DMSO concentration of 10.0 (v / v)%. This Ng-related peptide-containing sample solution was incubated at room temperature for 3 hours.

[0081] [Refining process] (First bonding step, first washing step, first dissolution step) A clone NG2 (manufactured by BioLegend) of the anti-Ng antibody (IgG1) with human neurogranin (Ng) residues 52-63 as its epitope was prepared. 100 μg of anti-Ng antibody was reacted with 5.5 mg of magnetic beads (Dynabeads M-270 Epoxy) in an immobilization buffer (0.1 M phosphate buffer containing 1.5 M ammonium sulfate; pH 7.4) at 37°C for 16-24 hours. This produced antibody beads, which served as antibody immobilization carriers.

[0082] The incubated Ng-related peptide-containing sample solution was allowed to stand on ice for 5-60 minutes, after which the antibody beads were mixed in and shaken on ice or at 4°C for 1 hour. The antibody beads were then washed 1 or 3 times with 100 μL of first washing buffer (0.05% UDM, 50 mM Tris-HCl, 150 mM NaCl; pH 7.4), and 1 or 2 times with 50 μL of 50 mM ammonium acetate buffer. Finally, the antibody beads were brought into contact with first acidic solution (0.05% UDM, 50 mM glycine buffer; pH 2.8) to elute the Ng-related peptide into the first acidic solution. This yielded a first eluate containing the Ng-related peptide.

[0083] (Neutralization process) The first eluate was mixed with a neutral buffer (0.1% UDM, 800 mM GlcNAc, 300 mM Tris-HCl, 300 mM NaCl; pH 7.4) to obtain a purified solution.

[0084] (Second bonding step, second washing step, second elution step) The purified solution was mixed with the antibody beads and shaken on ice or at 4°C for 1 hour. The antibody beads were then washed 2 or 5 times with 50 μL of second washing buffer (0.05% UDM, 50 mM Tris-HCl, 150 mM NaCl; pH 7.4), 1 or 2 times with 50 μL of 50 mM ammonium acetate buffer, and 1 time with 30 μL of water. The antibody beads were then contacted with a second acidic solution (70% (v / v) acetonitrile aqueous solution containing 5 mM hydrochloric acid and 0.1 mM methionine, 50 nM BSA) to elute the Ng-related peptide into the second acidic solution. This yielded a second eluate containing the Ng-related peptide.

[0085] [Detection process] A MALDI-TOF MS instrument, AXIMA Performance (Shimadzu / KRATOS, Manchester, UK), was used as the mass spectrometer. A 2 mg / mL CHCA / 0.2% (w / v) MDPNA matrix solution was prepared using α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix matrix, methylenediphosphonic acid (MDPNA) as the matrix additive, and acetonitrile as the solvent. 0.5 μL of the matrix solution was added dropwise to each of the four wells of a MALDI plate (μFocus MALDI plate 900 μm (Hudson Surface Technology, Inc., Fort Lee, NJ)), dried, and then 1 μL of the second eluate was added dropwise, followed by drying.

[0086] Next, MALDI-TOF MS was activated to detect ng-related peptides (SIL-Ng50-78, SIL-Ng50-75, rNg1-78, and rNg1-75). As a setting, mass spectral data was acquired using Linear TOF in positive ion mode. 400 spots and 16,000 shots were accumulated per well. The m / z values ​​of the Linear TOF were expressed as the average mass of the peaks. The m / z values ​​were calibrated using external standards: human angiotensin II, human ACTH fragments 18-39, bovine insulin oxidized beta-chain, bovine insulin, and cytochrome c.

[0087] Furthermore, if we were to target Ng50-78 and Ng50-75 that are not labeled with stable isotopes, and Ng1-78 and Ng1-75 that are not recombinant, it would become impossible to distinguish them from Ng50-78, Ng50-75, Ng1-78, and Ng1-75 that are naturally present in plasma, and the amount of degradation could not be accurately evaluated. Therefore, we targeted Ng50-78 and Ng50-75 that are not present in human plasma, and rNg1-78 and rNg1-75 with His tags.

[0088] Table 1 shows the Ng-related peptides detected by mass spectrometry. In the table, * indicates the theoretical average m / z of the divalent ion peak for rNg1-75 and rNg1-78.

[0089] [Table 1]

[0090] [Change in final concentration] The proportions of DMSO and buffer were changed to alter the DMSO concentration of the Ng-related peptide-containing sample solution obtained in the preparation step, ranging from 0 to 25.0 (v / v%) (specifically, 0%, 0.05%, 0.25%, 0.5%, 2.5%, 5.0%, 10.0%, 15.0%, 20.0%, and 25.0%). Otherwise, the preparation and detection steps were carried out in the same manner as described above.

[0091] These results are shown in Figures 1 to 6. Specifically, the mass spectra of Ng-related peptide-containing sample solutions at different DMSO concentrations are shown in Figure 1 (final concentration 0-5%) and Figure 2 (final concentration 0-30%), and graphs showing the relationship between the intensity ratio of the degraded peptide to the degraded peptide and the DMSO concentration are shown in Figures 3-4 (SIL-Ng50-75 / SIL-Ng50-78) and Figures 5-6 (rNg1-75 / rNg1-78).

[0092] <Comparative Example 1> The above preparation and detection steps were carried out in the same manner as described above, except that DMSO was not mixed in the preparation step (i.e., the DMSO concentration was set to 0%) and the incubation time was set to 0 minutes, 1 hour, and 3 hours, respectively.

[0093] These results are shown in Figures 7 to 9. Specifically, to demonstrate that Ng-related peptides collected from living organisms are degraded, Figure 7 shows the mass spectra at different incubation times when using a sample solution containing Ng-related peptides without DMSO, and Figures 8 (SIL-Ng50-75 / SIL-Ng50-78) and 9 (rNg1-75 / rNg1-78) show graphs illustrating the relationship between the intensity ratio of the degraded peptide to the degraded peptide and the incubation time.

[0094] <Consideration 1> (1) Decomposition of Ng in plasma when DMSO is not added. Figure 7 shows that the peak intensities of SIL-Ng50-75 and rNg1-75 increase with increasing incubation time, and Figures 8 and 9 show that the ratio of SIL-Ng50-75 to intact SIL-Ng50-78 and the ratio of rNg1-75 to intact rNg1-78 increase. This indicates that the C-terminal side of Ng75 is cleaved over time due to the influence of proteases in plasma.

[0095] (2) Inhibition of Ng degradation by DMSO addition Figures 1 to 6 show the analysis results after incubation of the sample solution with added DMSO for 3 hours. From Figures 1 and 2, the peaks for SIL-Ng50-75 and rNg1-75 become almost indistinguishable from the noise peaks in the DMSO concentration range of 5.0% or higher. Furthermore, from Figures 3 to 6, the ratio of SIL-Ng50-75 to intact SIL-Ng50-78, and the ratio of rNg1-75 to intact rNg1-78, decrease as the DMSO concentration increases, and are particularly low in the range of DMSO concentrations of 5.0% or higher. From this, it can be seen that the Ng decomposition inhibitory effect is sufficiently exerted when the DMSO concentration is 5.0% or higher. Furthermore, since the amounts of intact SIL-Ng50-78 and rNg1-78 stabilize (remain constant) over time, it can be seen that variations in these analytical results can be suppressed in mass spectrometry results for samples obtained from the same biological sample.

[0096] Furthermore, Figure 6 shows that when the DMSO concentration is 25.0% or higher, the ratio of rNg1-75 to intact rNg1-78 tends to increase. This is presumed to be because the excessive addition of DMSO alters the three-dimensional structure of rNg1-78, reducing antigen-antibody reactivity, and consequently decreasing the peak intensity of rNg1-78.

[0097] <Example 2> A sample solution containing Ng-related peptides was prepared in the same manner as in Example 1, except that acetone, dimethylformamide, ACN (acetonitrile), 2-propanol, or ethanol was used instead of DMSO, and the final concentration was 10.0%. The solution was incubated at room temperature for 3 hours. The purification and detection steps were then carried out in the same manner as in Example 1.

[0098] <Comparative Example 2> A sample solution containing Ng-related peptides was prepared in the same manner as in Example 1, except that methanol or toluene was used instead of DMSO and the final concentration was 10.0%, and the solution was incubated at room temperature for 3 hours. Then, the purification and detection steps were carried out in the same manner as in Example 1.

[0099] Graphs showing the intensity ratio of the degraded peptide to the degraded peptide at this time are shown in Figure 10 (SIL-Ng50-75 / SIL-Ng50-78) and Figure 11 (rNg1-75 / rNg1-78). The relative polarity of the organic solvents used in the examples and comparative examples is shown in Table 2.

[0100] [Table 2]

[0101] <Consideration 2> Figures 10 and 11 show that acetone, dimethylformamide, ACN, 2-propanol, and ethanol have an inhibitory effect on ng degradation, similar to DMSO. It is hypothesized that (1) nonpolar molecules hydrophobically interact with ng-degrading enzymes in plasma, blocking the hydrophobic interactions that support the three-dimensional structure of the ng-degrading enzymes and thus denaturing them, while (2) the polar groups of organic solvents form hydrogen bonds with the polar groups of ng-degrading enzymes, thereby denaturing them. Based on the balance of polarity and nonpolarity from (1) and (2), it is thought that organic solvents with a relative polarity within a certain range (0.200 or more, 0.700 or less) are particularly effective in denaturing ng-degrading enzymes and reducing ng degradation activity. However, the present invention is not limited to these hypothetical mechanisms.

[0102] <Example 3> Ng-related peptide-containing sample solutions were prepared in the same manner as in Example 1, except that ACN was mixed in place of DMSO, and the final concentrations were set to 0-30.0 (v / v%) (specifically, 0%, 0.05%, 0.25%, 0.5%, 2.5%, 5.0%, 10.0%, 15.0%, 20.0%, 25.0%, and 30.0%). These solutions were then incubated at room temperature for 3 hours. Subsequently, the purification and detection steps were carried out in the same manner as in Example 1.

[0103] The mass spectroscopy results for final concentrations of 0-20% are shown in Figures 12-14. The Ng-related peptides detected by mass spectroscopy are shown in Table 3. In the table, * indicates the theoretical average m / z of the divalent ion peak.

[0104] [Table 3]

[0105] <Consideration 3> Figures 12 to 14 show that when the ACN concentration is 10.0–20.0%, peaks for specific Ng peptides (Ng33-78, Ng45-68, Ng45-68, Ng48-78, Ng46-78, Ng45-78, Ng39-78, Ng33-78) are clearly observed, resulting in increased signal intensity and improved sensitivity. Furthermore, when the ACN concentration is 15.0–20.0%, in addition to the above, two divalent ion peaks for Ng46-78 and Ng39-78 are also observed, further improving sensitivity. On the other hand, when the ACN concentration is 5.0% or less and 25.0% or more, the above peaks were not clearly observed. Also, in the mass spectra of the organic solvents other than ACN (DMSO, acetone, dimethylformamide, ACN, 2-propanol, ethanol, methanol, toluene), the above peaks were not clearly observed.

[0106] The improved sensitivity of Ng-related peptides due to ACN addition is thought to be because ACN weakens hydrophobic interactions between proteins and peptides, making it easier to release Ng-related peptides that were adsorbed to proteins in plasma, and thus making it easier for antibodies to access the epitopes. However, the present invention is not limited to these hypothetical mechanisms.

Claims

1. A method for preparing a neurogranin-related peptide-containing sample solution, comprising mixing blood with an organic solvent having a relative polarity of 0.200 to 0.700 to prepare a sample solution in which the final concentration of the organic solvent is 5.0 (v / v) or more.

2. The preparation method according to claim 1, wherein the final concentration of the organic solvent is 5.0 (v / v)% or more and 20.0 (v / v)% or less.

3. The preparation method according to claim 1, further comprising mixing a buffer with the blood.

4. A preparation step that carries out the preparation method described in any one of claims 1 to 3, A measurement step in which mass spectrometry, liquid chromatography, immunoassay, or surface plasmon resonance is performed using the aforementioned sample solution. A method for analyzing neurogranin-related peptides, comprising the following features.

5. The measurement process described above is: (a) A binding step in which the sample solution is brought into contact with a carrier to obtain a conjugate in which the neurogranin-related peptide is bound to the carrier, (b) A cleaning step of cleaning the composite using a cleaning solution, (c) An elution step in which the conjugate is brought into contact with an acidic solution to obtain an eluate in which the neurogranin-related peptide has been eluted into the acidic solution, (d) A detection step of detecting the neurogranin-related peptide in the eluate by mass spectrometry. The analytical method according to claim 4, comprising:

6. The analytical method according to claim 5, wherein the ionization method of the mass spectrometry is matrix-assisted laser desorption ionization.

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